Koyanagi urethroplasty for proximal hypospadias: A stage procedure?

OBJECTIVE: To report experience and technical refinements with the Koyanagi urethroplasty for proximal hypospadias. METHODS: A retrospective study was conducted between 2004 and 2017. Medical records of patients treated by the Koyanagi technique were investigated. The penile ventral skin was closed either with a Byars flap or using an "Ombredanne's chasuble" (OC). The cohort was divided chronologically into two groups of the same number of patients (early -E- and late experience group-LEG-). Demographics, anatomical findings and surgical outcomes were compared between groups using univariate analysis. A logistic regression was performed to assess factors associated with the occurrence of a postoperative urethrocutaneous fistula or urethroplasty dehiscence (UD). RESULTS: The Koyanagi urethroplasty was performed in 67 patients, 33 in the EEG and 34 in the LEG. The overall surgical complication rate was 64.2% (n=43), including 42 fistula or UD, higher in the EEG (81.8%) than in the LEG (44.1%, P<0.01). However, in the LEG, patients underwent the surgery older and the use of OC more frequent. After multivariate analyses, factors associated with a postoperative fistula or UD were the year of surgery (OR=0.71 [0.53-0.96] P=0.02), the age at surgery (OR=1.11 [1.01-1.22], P=0.03); contrary to the skin coverage method or the stenting duration (P>0.05). An urethral stenosis occurred in 1 patient (1.5%). At last follow-up, 64.2% of patients required a further procedure and 80.6% of urethral meatus were glandular. CONCLUSION: In this study the complication rate, particularly the urethrocutaneous fistula, remained high. Urethral stenosis, were rare but the follow-up was too short to clearly identified them. Considering as a 2 stage procedure koyanagi uretroplasty allows to obtain finally good results. LEVEL OF EVIDENCE: III.

Image analysis for the automatic phenotyping of Orobanche cumana tubercles on sunflower roots.

BACKGROUND: The parasitic plant Orobanche cumana is one of the most important threats to sunflower crops in Europe. Resistant sunflower varieties have been developed, but new O. cumana races have evolved and have overcome introgressed resistance genes, leading to the recurrent need for new resistance methods. Screening for resistance requires the phenotyping of thousands of sunflower plants to various O. cumana races. Most phenotyping experiments have been performed in fields at the later stage of the interaction, requiring time and space. A rapid phenotyping screening method under controlled conditions would need less space and would allow screening for resistance of many sunflower genotypes. Our study proposes a phenotyping tool for the sunflower/O. cumana interaction under controlled conditions through image analysis for broomrape tubercle analysis at early stages of the interaction. RESULTS: We optimized the phenotyping of sunflower/O. cumana interactions by using rhizotrons (transparent Plexiglas boxes) in a growth chamber to control culture conditions and Orobanche inoculum. We used a Raspberry Pi computer with a picamera for acquiring images of inoculated sunflower roots 3 weeks post inoculation. We set up a macro using ImageJ free software for the automatic counting of the number of tubercles. This phenotyping tool was named RhizOSun. We evaluated five sunflower genotypes inoculated with two O. cumana races and showed that automatic counting of the number of tubercles using RhizOSun was highly correlated with manual time-consuming counting and could be efficiently used for screening sunflower genotypes at the tubercle stage. CONCLUSION: This method is rapid, accurate and low-cost. It allows rapid imaging of numerous rhizotrons over time, and it enables image tracking of all the data with time kinetics. This paves the way toward automatization of phenotyping in rhizotrons that could be used for other root phenotyping, such as symbiotic nodules on legumes.

Adaptative mechanism of the equilibrative nucleoside transporter 1 (ENT-1) and blood adenosine levels in elite freedivers.

PURPOSE: Long static or intense dynamic apnoea-like high-altitude exposure is inducing hypoxia. Adenosine is known to participate to the adaptive response to hypoxia leading to the control of heart rate, blood pressure and vasodilation. Extracellular adenosine level is controlled through the equilibrative nucleoside transporter 1 (ENT-1) and the enzyme adenosine deaminase (ADA). The aim of this study was to determine the control of adenosine blood level (ABL) via ENT-1 and ADA during apnoea-induced hypoxia in elite freedivers was similar to high-altitude adaptation. METHODS: Ten freediver champions and ten controls were studied. Biological (e.g. ENT-1, ADA, ABL, PaO2, PaCO2 and pH) and cardiovascular (e.g. heart rate, arterial pressure) parameters were measured at rest and after a submaximal dry static apnoea. RESULTS: In freedivers, ABL was higher than in control participants in basal condition and increased more in response to apnoea. Also, freedivers showed an ADA increased in response to apnoea. Finally, ENT-1 level and function were reduced for the free divers. CONCLUSION: Our results suggest in freedivers the presence of an adaptive mechanism similar to the one observed in human exposed to chronic hypoxia induced by high-altitude environment.

Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

[Latex-free hospitals: benefits and disadvantages]. / Hôpitaux pédiatriques sans latex: avantages et contraintes.

The objective of this article is to inform health-care personnel, especially those of pediatric hospitals (pediatricians, surgeons, anesthetists, etc.), about the incidence of latex allergy in children and the adverse effects that may result if avoidance measures are not taken. The prevalence of this allergy is increasing because of repeated exposure to this ubiquitous material (in medical equipment or household products such as bottle teats or balloons). The risks are allergic reactions, ranging from benign local dermatitis to anaphylactic shock. This problem is well known and progress has been made: there is increased production of latex-free products and screening for latex-related allergy or sensitization during the pre-anesthetic evaluation to take preventive actions when planning surgery. The younger the patient exposed to latex, the higher the risk of sensitization. Therefore, the main issue raised in this article is the potential benefit of moving to latex-free health-care facilities so as to minimize the risk of allergic incidents. Many studies showed a significant decrease in the risk of allergy (sensitization and allergic reaction) when avoiding latex, but we must also consider the feasibility, the cost, and the effectiveness of such a policy.

[Case report: neonatal delayed peristalsis after in-utero exposure to clozapine]. / Occlusion digestive néonatale fonctionnelle et transitoire après exposition in utero à la clozapine (Leponex(®)) : à propos d'un cas.

Schizophrenic women can now complete a pregnancy successfully, mostly due to psychiatric stabilization obtained with neuroleptic treatment. Side effects of classic neuroleptics are largely described in adults. On the other hand, effects of atypical neuroleptics, which are indicated when there is chronic severe schizophrenia with resistance or major intolerance to classic neuroleptics, are little known in newborn infants. We report the case of a hypertrophic full-term newborn whose mother received clozapine treatment alone with decreasing posology during the course of pregnancy. On his 2nd day of life, this newborn infant presented delayed peristalsis that required hospitalization in the digestive surgery department for more than 2 weeks. We assume that the anti-cholinergic effect of this molecule associated with a substantial plasmatic concentration and a possible increased half-life elimination were involved. Neonatal delayed peristalsis associated with clozapine treatment alone during pregnancy is poorly described in the medical literature. We only found one report on plasmatic concentrations of clozapine and its metabolite in a newborn, published in 1994. Close digestive monitoring is needed in the first days of life after in-utero exposure to clozapine and we believe that the question of a delayed initiation of enteral feeding after meconium elimination and in the absence of abdominal distension is debatable.

Priming of late endothelial progenitor cells with erythropoietin before transplantation requires the CD131 receptor subunit and enhances their angiogenic potential.

BACKGROUND: Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the ß-common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects. OBJECTIVE: The aim was to take advantage of the proangiogenic properties of EPO to enhance ECFC graft efficiency. We postulated that priming ECFCs by adding epoietin α in culture medium prior to experiments might increase their angiogenic properties. We also explored the role of the CD131 subunit in EPO priming of ECFCs. METHODS AND RESULTS: By western blotting on cord blood ECFC lysates, we showed that EPOR and CD131 expression increased significantly after EPO priming. These proteins coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFCs. EPO at 5 IU mL(-1) significantly stimulated proliferation, wound healing, migration and tube formation of ECFCs. EPO priming also increased ECFC resistance to H2 O2-induced apoptosis and survival in vivo. Similarly, in vivo studies showed that, as compared with non-primed ECFC injection, 5 IU mL(-1) EPO-primed ECFCs, injected intravenously 24 h after hindlimb ischemia in athymic nude mice, increased the ischemic/non-ischemic ratios of hindlimb blood flow and capillary density. These effects were all prevented by CD131 small interfering RNA transfection, and involved the phosphoinositide 3-kinase-Akt pathway. CONCLUSION: These results highlight the potential role of EPO-primed ECFCs for cell-based therapy in hindlimb ischemia, and underline the critical role of CD131 as an EPO coreceptor.

Soluble CD146 and APS: a potential biomarker of obstetrical complications?

Obstetric antiphospholipid syndrome (APS) seems to be a clinical subset of classical APS, for which the search for new markers is still challenging. Soluble CD146 (sCD146) constitutes a circulating endothelial biomarker. sCD146 is involved in the inflammatory response by promoting monocyte transmigration and displays chemotactic and angiogenic properties on endothelial cells. Its detection in human sera reveals physiological variations related to age and sex. A wide variation of sCD146 has been reported in several conditions. In particular, sCD146 levels are significantly higher in women presenting a history of recurrent fetal losses.

The second joint Kemali-IBRO Mediterranean school of neuroscience is the first for invertebrate neuroscience.

This meeting was held at the Stazione Zoologica Anton Dohrn, Naples, Italy, from 6th-13th July 2011. It was sponsored through a collaboration between IBRO (the International Brain Research Organisation) and the Kemali Foundation. Notably, it is the first IBRO sponsored School specifically in invertebrate neuroscience. The meeting was attended by early career researchers from countries around the Mediterranean basin and highlighted the research opportunities that this field can offer to neuroscientists at the start of their careers. Students participating in the School were introduced to a range of important invertebrate model systems. The utility of these models in addressing fundamental neurobiological problems, especially those relating to human neurological disorders, was extensively discussed. In this review, two of the participating students provide a summary of this meeting with a view to encouraging support for further activity in this arena in the future. One of the aims of the Kemali foundation is to foster enduring collaborations between scientists in the Mediterranean region. Given the enthusiastic networking both during and after the meeting, there is much promise that this goal will be realised.

[Free vascularized fibular flap in distal radius reconstruction]. / Transfert libre microvascularisé de fibula dans la reconstruction de l'extrémité inférieure du radius.

Free vascularized bone transfers are indicated to reconstruct large bone loss, either after traumatism or bone tumor resection. Free vascularized fibular transplant is particularly well designed for reconstruction of large defect of the distal radius. Anatomy is constant and harvesting is versatile and reliable. It may be used as bony transplant alone, osteocutaneous or osteocutaneomuscular. The length of the fibula harvested can be up to 25 cm. In children bone reconstruction, the proximal physis of the fibula can be transferred to allow secondary bone growth but there is a need for double anastomosis of the anteriotibial and the fibular bundles. Donor site sequelae are minimal.

Parametric and genetic analysis of Drosophila appetitive long-term memory and sugar motivation.

Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aversive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long-term memory (LTM), a long-lasting memory dependent on de novo protein synthesis. In contrast, one session of odour-sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative conditioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short-term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the unconditioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addition, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247-P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.

The early non-genomic aldosterone-induced increase in sodium transport is a membrane-initiated event that requires protein carboxyl methylation in renal cells.

Effects of aldosterone on its target cells are generally considered to be mediated through the genomic pathway. However, recent studies have evidenced rapid effects of the hormone that involve a non-genomic mechanism. In this study, we show that, in the RCCD2 rat cortical collecting duct cell line, the early effect of the hormone on transepithelial sodium transport is neither antagonized by the mineralo- and glucocorticoid receptors antagonists RU26752 and RU486, nor blocked by mRNA and protein synthesis inhibitors. Interestingly, the plasma membranes of RCCD2 cells specifically bind 3H-aldosterone but not 3H-dexamethasone, a binding that is not displaced in the presence of RU26752 or RU486, suggesting the presence of an aldosterone membrane receptor. In addition, the early aldosterone-induced increase in sodium transport is blocked by the addition of a specific inhibitor of carboxyl methyl transferase. These results suggest that, in RCCD2 cells, the early aldosterone-induced increase in sodium transport is not mediated through the genomic pathway but through a membrane receptor-mediated signal and could involve a rapid carboxyl methylation process regulated by aldosterone.

Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1.

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter- gus/ gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4-5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T(1) descendents.

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line.

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.

A novel model for equine recurrent airway obstruction.

Equine recurrent airway obstruction (RAO; a term combining both chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD)) is one of the most common equine respiratory diseases with up to 50% of horses affected worldwide. The etiopathogenesis of RAO is unknown although pulmonary hypersensitivity to inhaled mold antigens may be involved. Recent work in our laboratory demonstrating elevated levels of IL-4 and IL-13 mRNA in the airways and peripheral blood of horses with RAO is consistent with an atopic component to RAO. Little is known regarding the earliest phases of RAO in horses. Here we describe the development of a novel airway model for equine RAO that utilizes ovalbumin-coated polystyrene beads for airway sensitization and challenge. Aerosol challenge of sensitized ponies with OVA-coated microbeads resulted in decreased airway compliance, increased percentage of lymphocytes and neutrophils in the bronchoalveolar lavage fluid, and evidence of a Th2 cytokine response in the bronchoalveolar cells. These results suggest that this approach may be useful in describing the initial stages of RAO development in the horse.

Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi.

• An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. • Agrobacterium rhizogenes transformed root explants were derived either from Myc + M. truncatula or from the infection-defective Myc - mutant TR26 ( dmi2-2 ), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. • Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc + roots, but not in epidermal cells in contact with appressoria in Myc - roots. • We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.

Tetracycline-inducible gene expression in cultured rat renal CD cells and in intact CD from transgenic mice.

The renal collecting duct (CD) plays a key role in the control of ion and fluid homeostasis. Several genetic diseases that involve mutations in genes encoding for ion transporters or hormone receptors specifically expressed in CD have been described. Suitable cellular or transgenic animal models expressing such mutated genes in an inducible manner should represent attractive systems for structure-function relationship analyses and the generation of appropriate physiopathological models of related diseases. Our first goal was to develop a CD cell line that allows inducible gene expression using the tetracycline-inducible system (Tet-On). We designed several strategies aimed at the development of a tight and highly inducible system in RCCD1 cells, a rat cortical collecting duct (CCD) cell line exhibiting several properties of the native CCD. Analysis of reporter gene expression demonstrated that the Tet-On system is suitable for inducible gene expression in these cells. In a second step, we have tested whether transgenic Tet-On mice expressing the tetracycline transactivator under the control of the human cytomegalovirus promoter were suitable for inducible gene expression in tubule epithelial cells. The results indicate that, in vivo, the inducible expression of the lacZ reporter gene appeared to be restricted to the CD. This particular strain of transgenic mice may therefore be useful for the expression of genes of interest in an inducible manner in the collecting duct.

Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines.

Macrophage inflammatory protein (MIP)-3alpha is a chemokine involved in the migration of T cells and immature dendritic cells. To study the contribution of proinflammatory cytokines and chemokines to the recruitment of these cells in rheumatoid arthritis (RA) synovium, we looked at the effects of the monocyte-derived cytokines IL-1beta and TNF-alpha and the T cell-derived cytokine IL-17 on MIP-3alpha production by RA synoviocytes. Addition of IL-1beta, IL-17, and TNF-alpha induced MIP-3alpha production in a dose-dependent manner. At optimal concentrations, IL-1beta (100 pg/ml) was much more potent than IL-17 (100 ng/ml) and TNF-alpha (100 ng/ml). When combined at lower concentrations, a synergistic effect was observed. Conversely, the anti-inflammatory cytokines IL-4 and IL-13 inhibited MIP-3alpha production by activated synoviocytes, but IL-10 had no effect. Synovium explants produced higher levels of MIP-3alpha in RA than osteoarthritis synovium. MIP-3alpha-producing cells were located in the lining layer and perivascular infiltrates in close association with CD1a immature dendritic cells. Addition of exogenous IL-17 or IL-1beta to synovium explants increased MIP-3alpha production. Conversely, specific soluble receptors for IL-1beta, IL-17, and TNF-alpha inhibited MIP-3alpha production to various degrees, but 95% inhibition was obtained only when the three receptors were combined. Similar optimal inhibition was also obtained with IL-4, but IL-13 and IL-10 were less active. These findings indicate that interactions between monocyte and Th1 cell-derived cytokines contribute to the recruitment of T cells and dendritic cells by enhancing the production of MIP-3alpha by synoviocytes. The inhibitory effect observed with cytokine-specific inhibitors and Th2 cytokines may have therapeutic applications.

Hypoxia reduces alveolar epithelial sodium and fluid transport in rats: reversal by beta-adrenergic agonist treatment.

In cultured alveolar epithelial cells, hypoxia induces a downregulation of the two main Na proteins, the epithelial Na channel (ENaC) and the Na,K-ATPase. However, the in vivo effects of hypoxia on alveolar epithelial transport have not been well studied. Therefore, the objectives of this study were to investigate in an in vivo rat model if hypoxia induces a reduction in vectorial Na and fluid transport across the alveolar epithelium in vivo, and if a change in net fluid transport is associated with modification in the expression and/or activity of Na transport proteins. Rats were exposed to 8% O(2) from 3 to 24 h. Hypoxia induced a progressive decrease in alveolar liquid clearance (ALC) reaching 50% at 24 h, an effect that was related primarily to a decrease in amiloride-sensitive transepithelial Na transport. On RNase protection assay of alveolar type II (ATII) cells isolated immediately after hypoxic exposure, steady state levels of mRNA were increased for alpha-rENaC and beta(1)-Na, K-ATPase, whereas the levels of gamma-rENaC and alpha(1)-Na,K-ATPase were unchanged. On Western blots of ATII cell membranes, alpha-ENaC subunit protein slightly increased, whereas the amount of alpha(1)- and beta(1)-Na,K-ATPase protein were unchanged with hypoxia. Thus, the decrease in transepithelial Na transport was not explained by a parallel change in gene expression or the quantity of transport proteins. Interestingly, hypoxia-induced decrease in ALC was completely reversed by intra-alveolar administration of the beta(2) agonist, terbutaline (10(-4) M). These results suggest that hypoxia-induced decrease in Na transport is not simply related to a downregulation of Na transport proteins but rather to a decrease in Na protein activity by either internalization of the proteins and/or direct alteration of the protein in the membrane. The dramatic increase of ALC with beta(2)-agonist therapy indicates that the decrease of transepithelial Na and fluid transport during hypoxia is rapidly reversible, a finding of major clinical significance.